Discovery of photosynthesis genes through whole-genome sequencing of acetate-requiring mutants of Chlamydomonas reinhardtii

摘要

Large-scale mutant libraries have been indispensable for genetic studies, and the development of next-generation genome sequencing technologies has greatly advanced efforts to analyze mutants. In this work, we sequenced the genomes of 660 Chlamydomonas reinhardtii acetate-requiring mutants, part of a larger photosynthesis mutant collection previously generated by insertional mutagenesis with a linearized plasmid. We identified 554 insertion events from 509 mutants by mapping the plasmid insertion sites through paired-end sequences, in which one end aligned to the plasmid and the other to a chromosomal location. Nearly all (96%) of the events were associated with deletions, duplications, or more complex rearrangements of genomic DNA at the sites of plasmid insertion, and together with deletions that were unassociated with a plasmid insertion, 1470 genes were identified to be affected. Functional annotations of these genes were enriched in those related to photosynthesis, signaling, and tetrapyrrole synthesis as would be expected from a library enriched for photosynthesis mutants. Systematic manual analysis of the disrupted genes for each mutant generated a list of 253 higher-confidence candidate photosynthesis genes, and we experimentally validated two genes that are essential for photoautotrophic growth, CrLPA3 and CrPSBP4 . The inventory of candidate genes includes 53 genes from a phylogenomically defined set of conserved genes in green algae and plants. Altogether, 70 candidate genes encode proteins with previously characterized functions in photosynthesis in Chlamydomonas , land plants, and/or cyanobacteria; 14 genes encode proteins previously shown to have functions unrelated to photosynthesis. Among the remaining 169 uncharacterized genes, 38 genes encode proteins without any functional annotation, signifying that our results connect a function related to photosynthesis to these previously unknown proteins. This mutant library, with genome sequences that reveal the molecular extent of the chromosomal lesions and resulting higher-confidence candidate genes, will aid in advancing gene discovery and protein functional analysis in photosynthesis. Author summary Oxygenic photosynthesis in plants, algae, and cyanobacteria is responsible for the production of organic matter and oxygen that supports most life on Earth, yet we still do not fully understand all the genes that are necessary for this fundamental biological process. Forward genetics is an unbiased approach to finding genes that are important for a biological process of interest. To identify genes that are necessary for oxygenic photosynthesis in the unicellular green alga Chlamydomonas reinhardtii , we sequenced the genomes of 660 mutants that are defective in photosynthesis. By manual curation of each mutant and its disrupted gene(s) and cross-comparison with previous photosynthesis mutant studies, we have identified 253 genes in 328 mutants that are strong candidate genes for having roles in photosynthesis. The list of 253 genes consists of 70 known photosynthesis genes, 169 previously uncharacterized genes, and 14 genes previously characterized for functions unrelated to photosynthesis. In addition, we gained insight into the molecular events that accompany insertional mutagenesis in Chlamydomonas , such as large deletions and chromosomal rearrangements. Our mutant library with documented mutant phenotypes, sequenced genomes, and candidate genes will aid in the discovery of photosynthesis genes.